Polymer-based well plate devices and fluidic systems and methods of making and using the same

ABSTRACT

Embodiments of substrate-based devices disclosed herein comprise a polymeric substrate with a hydrophobic polymer coating that defines wells or openings on the polymeric substrate and/or serves as an adhesive agent between stacked substrates of the device. Also disclosed herein are embodiments of plate frames that can be used to align and hold well plate devices described herein. Methods of fabricating the disclosed devices also are described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/516,620, filed Jun. 7, 2017, which is incorporatedherein by reference in its entirety.

FIELD

The present disclosure concerns polymer-based devices and microfluidicsystems useful for diagnostic and analytical methods and methods ofmaking and using the same.

BACKGROUND

Microfluidic paper-based analytical devices (μPADS) have increased inpopularity as a means of decreasing the cost of common clinicalchemistry assays and making these tests available to the developingworld since the mid 2000's. There is currently no standard means ofdeveloping/modifying the assay chemistry, or detecting the resultingoutput (colorimetric or fluorescence) of such devices. Similarly, thereis a large segment of the developed world that can make use of alow-cost alternative to expensive well plates and manual processes foradding reagents commonly used for laboratory based assays. Thus, a needin the art exists for devices, and methods for fabricating such devices,that provide a low-cost alternative to conventional expensive devicesused for diagnostic and/or analytical techniques.

SUMMARY

Disclosed herein are embodiments of a polymer-based analytical device.In some embodiments, the device comprises a substrate comprising acoating of a hydrophobic polymer component wherein the coating of thehydrophobic polymer is configured to define outer perimeters of wells onor openings in the substrate and wherein the hydrophobic polymercomponent has a structure satisfying Formula I

wherein Z, Y, and W independently can be O, S, NH, or NR², where R² ishydrogen, aliphatic, aryl, or heteroaryl; each of R³, R⁴, R⁵ and R⁶ (ifpresent) independently are hydrogen, aliphatic, aryl, heteroaryl, or aheteroatom-containing moiety; r is an integer selected from 1 to 4; sand t independently are integers selected from 0 to 4; and q is aninteger selected from 1 to 1000.

In yet additional embodiments, the devices can comprise a bottomsubstrate comprising a coating of a hydrophobic polymer component; anintermediate substrate coupled to the bottom substrate, the intermediatesubstrate comprising a coating of a hydrophobic polymer component thatdefines outer perimeters of wells on or openings in the intermediatesubstrate; a sensor substrate coupled to the intermediate substrate, thesensor substrate comprising a signaling moiety or a sample; and a topsubstrate coupled to the sensor substrate, the top substrate comprisinga coating of a hydrophobic polymer component that defines outerperimeters of wells or openings having a pattern matching a pattern ofthe wells or openings of the intermediate substrate; wherein thehydrophobic polymer component has a structure satisfying Formula I:

wherein Z, Y, and W independently can be O, S, NH, or NR², where R² ishydrogen, aliphatic, aryl, or heteroaryl; each of R³, R⁴, R⁵ and R⁶ (ifpresent) independently are hydrogen, aliphatic, aryl, heteroaryl, or aheteroatom-containing moiety; r is an integer selected from 1 to 4; sand t independently are integers selected from 0 to 4; and q is aninteger selected from 1 to 1000.

Also disclosed herein are embodiments of methods of making polymer-basedanalytical devices. In some embodiments, the methods can comprisemasking a substrate made of a polymeric material with a masking materialto form a masked substrate; patterning the masked substrate by cutting apre-determined pattern into the masking material thereby providinghydrophilic unmasked areas of the masked substrate and masked areas ofthe masked substrate; coating the hydrophilic unmasked areas of thesubstrate with a hydrophobic polymer layer thereby converting thehydrophilic unmasked areas of the masked substrate to hydrophobicunmasked areas; and removing any remaining masking material from themasked substrate to expose one or more wells or openings, each of whichhas an outer perimeter defined by the hydrophobic polymer layer of thesubstrate and wherein the wells or openings are configured in thepre-determined pattern.

In yet additional embodiments, the methods can comprise exposing apolymeric substrate to a solution of a hydrophobic polymer component toform a layer of the hydrophobic polymer component that fully covers thepolymeric substrate thereby forming a fully-coated polymeric substrate;patterning the fully-coated polymeric substrate to comprise a pluralityof wells or openings thereby forming a patterned polymeric substrate,wherein each well or opening of the plurality of wells or openings hasan outer perimeter defined by the hydrophobic polymer; exposing thepatterned polymeric substrate to O₂ to render the hydrophobic polymerlocated in the wells or openings; and coupling the patterned polymericsubstrate with a hydrophobic polymer-coated bottom substrate.

Also disclosed herein are embodiments of a plate frame. In someembodiments, the plate frame can comprise a first component made of apolymeric material, such as a biodegradable polymer, and having an outerperimeter section; a second component made of a polymeric material, suchas a biodegradable polymer, and having an outer perimeter sectionconfigured to align with the outer perimeter section of the firstcomponent; one or more magnets positioned within the outer perimetersection of the second component; and one or more magnets positionedwithin the outer perimeter section of the first component, which magnetsof the first component are positioned to align with the magnets of thesecond component and secure the first component to the second componentin a predetermined alignment by magnetic attraction; wherein when thefirst component and the second component are associated together, theydefine a chamber that is configured to receive a polymer-basedanalytical device as described herein between the second component andthe first component.

The foregoing and other objects, features, and advantages of the presentdisclosure will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded perspective view of a representative paper-basedstrip device.

FIG. 2 is an exploded perspective view of a representative paper-based,96-well plate device.

FIG. 3 is an exploded perspective view of a representative paper-based,36-well plate device.

FIG. 4 is an image showing a top view of an assembled paper-based,96-well plate device.

FIG. 5 is an exploded perspective view of a representative paper-based,96-well plate device with transparent bottom layer such that anygenerated signal produced during use of the well plate device can bedetected either from viewing the well plate device from the top orbottom.

FIG. 6 is an exploded perspective view of a representative paper-based96-well plate device comprising fluidic channels between the openings ofthe intermediate substrate component of the device.

FIG. 7 is an exploded perspective view of a paper-based microfluidicwell plate with multiple openings connected together allowing formultistep assays or multiple tests from a single sample to be conductedusing the device.

FIG. 8 is top view image of a microfluidic well plate having openingsconnected with fluidic channels whereby different dyed fluids are usedto illustrate the interconnected nature of the openings.

FIG. 9 is an exploded perspective view of a plate frame deviceembodiment comprising first and second components having magnets.

FIG. 10 is an exploded perspective view of a representative plate framedevice.

FIGS. 11A and 11B are images of components of a constructed magneticplate frame device.

FIG. 12 is an illustration of a representative alignment device thatfriction fits a second portion of a plate frame device thus facilitatingwell plate substrate alignment upon addition of the magnetic firstportion of the frame device.

FIGS. 13A-13D are photographic images showing positioning and use of arepresentative alignment device (FIG. 13A) to align well platesubstrates (FIG. 13C) and the first and second components of a plateframe device (FIGS. 13B and 13D).

FIGS. 14A-14C are illustrations of a representative device embodimentdescribed herein, wherein FIG. 14A illustrates an intermediate substratecomprising three rows of wells connected via fluidic channels; FIG. 14Billustrates an embodiment wherein the intermediate substrate of FIG. 14Ahas been coupled with a top substrate that facilitates visualization ofthe wells of the intermediate substrate; and FIG. 14C illustrates anembodiment wherein the intermediate substrate of FIG. 14A has beencoupled with a top substrate that facilitates visualization of the wellsand fluidic channels of the intermediate substrate.

FIGS. 15A-15C are images of a representative device as illustrated inFIGS. 14A-14C after being treated with dye-containing samples tovisualize fluid flow through the wells and fluidic channels of thedevice; FIG. 15A shows an intermediate substrate after deposition of ared dye into a well of the intermediate substrate; FIG. 15B shows anintermediate substrate coupled with a top substrate that allowsvisualization of the wells of the intermediate substrate and wherein itcan be seen that fluid flow through the wells and fluidic channels ofthe device allows the blue dye shown in the well of column b, row 2 tobe transferred to the well of column c, row 3 by red dye introduced intothe well of column a, row 3.

FIGS. 16A and 16B illustrate another representative device comprising aplurality of stacked substrates, wherein FIG. 16A illustrates a top planview of the fully constructed device and FIG. 16B illustrates anexploded top view of the device of FIG. 16A, and shows the differentsubstrates that make-up the device.

FIG. 17 is a schematic illustration of a method for using asubstrate-based analytical device described herein in a cyanidedetection method.

FIGS. 18A-18C illustrate characterization data of chitosan encapsulatedCdTe QDs; FIG. 18A shows emission spectra of CS-QD520 compared to theoriginal QD520 (where the inset shows image “a,” which is CS-QD520 underUV light at 365 nm and image “b,” which is QD520 under UV light 365 nm;FIG. 18B is a TEM image of CS-QD520; and FIG. 18C is a ChemiSTEM modeimage of the same area of FIG. 18B showing elemental analysis.

FIGS. 19A and 19B are images showing the interaction of CS-QD520 on asensor substrate surface wherein both QD520 and CS-QD520 were applied onthe sensor substrate surface and dried in the vacuum oven; FIG. 19Aillustrates results before flushing with PB buffer 10 mM, pH 7; and FIG.19B shows results after flushing with PB buffer 10 mM, pH 7.

FIGS. 20A and 20B are graphs showing selectivity of a representativeassay on paper-based well plates; FIG. 20A illustrates results from aset of tested anions at 1 mM compared to cyanide at 100 μM; and FIG. 20Billustrates results from a set of tested cations at 1 mM compared tocyanide at 100 μM.

FIGS. 21A and 21B show the comparative sensitivity of the assay in asolution (FIG. 21A) and on a paper-based well plate (FIG. 21B) with thefollowing conditions: CS-QD520 at 8.17 μM was quenched with 50 μL-Cu²⁺(100 mg/L) for 2 hours; wherein for the solution-based assay, 50 μL ofsensor was mixed with 50 μL of cyanide standard.

FIGS. 22A-22C are graphs showing optimization parameters for cyanidedetection on paper-based well plates; FIG. 22A shows the effect ofCS-QD520 concentration [conditions: ratio of CSQD: Cu²⁺ (100 mg/L)=4.085μM: 25 μL, reaction time=30 min]; FIG. 22B shows the effect of amount ofCu²⁺ (100 mg/L) for quenching a CS-QD520 probe [conditions: CSQD=8.17μM, reaction time=30 min]; and FIG. 22C shows the effect of reactiontime [conditions: CSQD=8.17 μM, Cu²+, 100 mg/L=40 μL].

FIG. 23 is an endpoint calibration graph derived from paper-based wellplates for cyanide detection wherein the following conditions were used:CS-QD520=8.17 μM, amount of Cu²⁺ (100 mg/L)=40 μL/1 mL of 8.17 μMCS-QD520, reaction time=30 min.

FIG. 24 is an image of a representative well plate device wherein thewell plate device is made using aerosolized deposition of PCL whereinmasking tape is used to cover the areas of desired hydrophilicity tothereby form wells on the substrate.

FIGS. 25A and 25B are images of a representative well plate device (FIG.25A) and a corresponding calibration curve for glucose data obtainedfrom using the device (FIG. 25B), wherein uric acid, glucose, andbilirubin (×2) assays (left to right) were conducted using the device.

FIGS. 26A and 26B are photographic images of additional well platedevices formed by filling filter paper with PCL via soaking and whereinthe openings are cut using a laser cutter or another method and multiplelayers are laminated together; FIG. 26A shows the device and FIG. 26Bshows the device when placed within a plate frame.

FIGS. 27A-7E are photographic images showing a representative low volume“dimple” plates comprising a polymer film (e.g.,polyethyleneteraphthalate [“PET” ]) sprayed with a polymer component(e.g., PCL) to provide adhesion between laminated substrates; FIG. 7A isa top view of a constructed well plate device; FIG. 27B is a top view ofa constructed well plate device in a plate frame; FIG. 27C is aphotographic image of the device after colored fluids have been added toshow the ability of the well plate to hold and separate the fluids; FIG.27D is a photographic image of the device placed on white background;FIG. 7E is a zoomed photographic image of the device.

FIG. 28 is an illustration of a multilayer device which includes asingle-use valve system that isolates or connects adjacent wells.

FIGS. 29A and 29B are cross-sectional illustrations of valve embodimentsdescribing their operation (normally open and normally closed valveactuation mechanisms, respectively.)

FIG. 30 is an image of a representative device that comprises anintermediate substrate having wells passivated with bovine serum albuminso as to prevent interaction between areas of the well and the depositedblue dye.

FIG. 31 is an image obtained from fluorescence image of a deviceembodiment wherein one well has been passivated with bovine serumalbumin (left), one well has not been passivated (middle); and one wellhas been treated with trimethyl chlorosilane (right).

DETAILED DESCRIPTION I. Explanation of Terms

The following explanations of terms are provided to better describe thepresent disclosure and to guide those of ordinary skill in the art inthe practice of the present disclosure. As used herein, “comprising”means “including” and the singular forms “a” or “an” or “the” includeplural references unless the context clearly dictates otherwise. Theterm “or” refers to a single element of stated alternative elements or acombination of two or more elements, unless the context clearlyindicates otherwise.

Any theories of operation are to facilitate explanation, but thedisclosed devices, materials, and methods are not limited to suchtheories of operation. Although the operations of some of the disclosedmethods are described in a particular, sequential order for convenientpresentation, it will be understood that this manner of descriptionencompasses rearrangement, unless a particular ordering is required byspecific language set forth below. For example, operations describedsequentially may in some cases be rearranged or performed concurrently.Moreover, for the sake of simplicity, the attached figures may not showthe various ways in which the disclosed components and materials can beused in conjunction with other components and materials. Additionally,the description sometimes uses terms such as “produce” and “provide” todescribe the disclosed methods. These terms are high-level abstractionsof the actual operations that are performed. The actual operations thatcorrespond to these terms will vary depending on the particularimplementation and are readily discernible by one of ordinary skill inthe art.

In some examples, values, procedures, or devices are referred to as“lowest,” “best,” “minimum,” or the like. It will be appreciated thatsuch descriptions are intended to indicate that a selection among manyused functional alternatives can be made, and such selections need notbe better, smaller, or otherwise preferable to other selections.

Examples are described with reference to directions indicated as“above,” “below,” “upper,” “lower,” and the like. These terms are usedfor convenient description, but do not imply any particular spatialorientation.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting, unless otherwiseindicated. Other features of the disclosure are apparent from thefollowing detailed description and the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, percentages, temperatures, times, and soforth, as used in the specification or claims are to be understood asbeing modified by the term “about.” Accordingly, unless otherwiseindicated, implicitly or explicitly, the numerical parameters set forthare approximations that can depend on the desired properties soughtand/or limits of detection under standard test conditions/methods. Whendirectly and explicitly distinguishing embodiments from discussed priorart, the embodiment numbers are not approximates unless the word “about”is recited. Furthermore, not all alternatives recited herein areequivalents.

To facilitate review of the various embodiments of the disclosure, thefollowing explanations of specific terms are provided:

Cellulosic Polymer: A polymer made of cellulose or a derivative thereof.

Elastomeric Polymer: A flexible polymer. Exemplary elastomeric polymersinclude, but are not limited to, unsaturated rubbers, such aspolyisoprene or polybutadiene, and saturated rubbers, such asepichlorohydrin and ethylene-vinyl acetate.

Opening: An aperture or gap in the surface of a substrate that allows afluid or solid to pass through the substrate and/or that serves tocontain a fluid or solid within a perimeter of the opening. In someembodiments, openings are surrounded by a hydrophobic polymer componentand thus the hydrophobic polymer defines the outer perimeter of theopening.

Sump: A region of a substrate used in certain device embodiments that isconfigured to accept a volume of a fluid, such as an aqueous solution,melted polymer and/or wax used as a valve in device embodimentsdescribed herein.

Synthetic Fiber Polymer: A polymer that is not found in nature, butmimics physical and/or chemical properties of a natural plant or animalfiber.

Thermoplastic Polymer: A type of polymer that becomes moldable andmalleable above a particular temperature, and that solidifies uponcooling. Exemplary thermoplastic polymers include, but are not limitedto, polyamides, polylactic acid, polycarbonate, polyetherimide,polypropylene, polystyrene, polyvinyl chloride, polytetrafluoroethylene,and the like.

Thermoset Polymer: A type of polymer that changes from a soft or viscousstate into a hard polymer by curing and does not change shape aftercuring. Exemplary thermoset polymers include, but are not limited to,polyester resin, polyurethanes, epoxy resin, cyanate esters, and thelike.

Well: A region of a substrate used in certain device embodimentsdescribed herein wherein the region is hydrophilic and thus capable ofabsorbing a fluid, such as a liquid. In some embodiments, a well isdefined on the surface of a polymeric substrate by a hydrophobic polymerlayer. In other embodiments, a well is defined as a region of asubstrate that is hydrophilic and not filled with a hydrophobic flowbarrier (polymer), which is surrounded by that same substrate which hasbeen modified by deposition of a hydrophobic or less hydrophilicmaterial such as a polymer.

II. Introduction

The substrate-based devices described herein are biodegradable,disposable devices that provide an inexpensive alternative toconventional expensive devices used in analytical and biochemicalanalysis, such as polymeric well plates (or micro-titer plates). In someembodiments, the disclosed devices include paper-based devices, such asanalytical paper-based strips and/or paper-based well plates. Thedisclosed devices also can utilize a separate polymeric material (thatis, a polymeric material that is separate from the polymer material ofthe substrates of the substrate-based devices) as a device component andas a bonding agent capable of adhering device substrates together.

The disclosed devices can be used to avoid multi-step assays as thedevices provide a mechanism for combining different steps of amulti-step assay into a single step by incorporating a microfluidicarchitecture that connects multiple wells and/or openings of a wellplate and/or stacking layers that can provide a plurality of reagents.This allows for reagents to be stored separately on a well plate, or tobe added via robotic liquid handling (or even manually) thus eliminatingor reducing washing steps necessary in traditional multi-step assaysconducted in well plates. The devices also provide the possibility ofincluding reagents in dried form into the disposable, low-cost wellplate assay device. The devices disclosed herein can be integrated withcomponents typically used for analytical analysis, such as platereaders, plate washers, plate stackers/handlers, and dispensingtechnologies.

III. Devices

Disclosed herein are embodiments of substrate-based analytical devices.In some embodiments, the devices are polymeric substrate-basedanalytical devices suitable for fluid handling in analytical techniques,such as analyte detection, qualification, or quantification,diagnostics, biological assays, and the like. The disclosed devices aresubstrate-based devices that comprise a patterned substrate made of apolymeric material and/or a fibrous material; or a plurality of stackedsubstrates, each made of a polymeric material and/or a fibrous material.In some embodiments, the polymeric material is a cellulosic polymer andthe fibrous material is a glass fiber material. In some otherembodiments, the polymeric material is a thermoplastic polymer, athermoset polymer, an elastomeric polymer, a synthetic fiber polymer, ora combination thereof. In some embodiments, the fibrous material is aglass microfiber material. In particular disclosed embodiments, thesubstrates are made from a porous polymeric material, such as a materialmade with a cellulose (or cellulose derivative) pulp; or a polymericmaterial, such as polyethyleneteraphthalate (PET). In some embodiments,the substrates can be made using a material that is suitable for use inUV-Vis analysis or fluorescence analysis. Materials suitable for use influorescence analysis typically have a dark color (e.g., black) suchthat it does not fluoresce using fluorescent analytical techniques.

In particular disclosed embodiments, the disclosed substrate-basedanalytical devices include substrate-based strip devices andsubstrate-based well plate components (e.g., substrate-based well platesand well plate frames). The disclosed substrate-based analytical devicesof the present disclosure utilize a biodegradable hydrophobic polymer toprovide a hydrophobic barrier on certain substrates used for thesubstrate-based analytical device. In particular disclosed embodiments,the hydrophobic polymer is used to define wells of a substrate. Forexample, the substrate-based analytical devices can be paper-baseddevices that comprise paper strips patterned with a single row of wellsused for strip-based devices, wherein the paper is coated with thehydrophobic polymer such that it defines the walls of the wells. In yetadditional embodiments, the paper strips are patterned with a single rowof openings wherein the outer perimeter of each opening is surrounded bythe hydrophobic polymer. In additional embodiments, the hydrophobicpolymer can be used to make substrates that can act as an adhesive toconnect different layers of a device. In additional embodiments, thesubstrate-based analytical devices are paper-based well plate devicesthat comprise paper substrates patterned with a plurality of wellswherein the outer perimeter of each well is defined by the hydrophobicpolymer. In yet some additional embodiments, the hydrophobic polymer canprovide a hydrophobic barrier between openings of a substrate. Polymericsubstrates also are contemplated wherein the strip is a strip ofpolymeric (e.g., PET) material or wherein the well plate substrates arepolymeric (e.g., PET) substrates.

In particular disclosed embodiments, the substrate-based analyticaldevices comprise at least one patterned substrate and can furthercomprise one or more additional substrates that may or may not bepatterned. Each substrate of the substrate-based analytical device canbe modified with the hydrophobic polymer such that the polymercompletely covers the substrate (e.g., the hydrophobic polymer coversfrom 95% to 100% of the surface area of one or all surfaces of thesubstrate) or partially covers the substrate (e.g., the hydrophobicpolymer covers less than 100% of the surface area of one or all surfacesof the substrate, such as 1% to 95%, or 5% to 90%, or 10% to 80%). Inyet additional embodiments, the polymer can cover the substrate only inareas defining wells and/or openings of the substrate such that thepolymer is not located within the wells or the openings. In yetadditional embodiments, the polymer does not cover openings of asubstrate.

In particular disclosed embodiments, the substrate-based analyticaldevice can comprise multiple substrates. For example, some embodimentscomprise a base substrate that typically is a non-patterned substrate;one or more intermediate substrates that may or may not be patterned tocomprise a plurality of wells and/or a plurality of openings; a topsubstrate that typically is, but need not be, a patterned substratecomprising a plurality of wells and/or a plurality of openings; and anycombination thereof. In particular disclosed embodiments, the devicescomprise a bottom substrate, an intermediate substrate, and a topsubstrate.

In particular disclosed embodiments, the top substrate is patterned witha plurality of wells and/or openings that are patterned in aconfiguration matching a pattern of wells and/or openings formed in anintermediate substrate. In yet additional embodiments, the wells and/oropenings patterned in the top substrate can be configured to be smallerthan the wells and/or openings patterned in the intermediate substrate.In yet additional embodiments, the top substrate does not need to bepatterned to comprise wells or openings and instead can be used toprotect an immobilized reagent from leaking from other substrates of thedevice and/or protect such reagents from degradation (e.g.,photodegradation) and possible contamination or side reactions (e.g.,oxidation). The wells and/or openings of both the top and intermediatesubstrates can have any shape, such as circular, rectangular, square,oval, or any other geometrical shape. In representative embodiments, thewells and/or openings of the top and intermediate substrates arecircular. In some embodiments, the wells of the top and/or intermediatesubstrate (or even sensor substrates described herein) can be modifiedto comprise a surface-passivating agent, such as a blocking agent (e.g.,bovine serum albumin, milk protein, or the like), or a silylating agent,such as trimethyl chlorosilane (TMCS), or other silicon-containingreagents. These surface-passivating agents can be used to preserve assaychemistry and or to enhance output signals produced when using thedevice.

The wells and/or openings of both the top and intermediate substratesalso can be configured to have a particular size that can be selected tomatch the particular methods for which the substrate-based analyticaldevice is to be used. In some embodiments, the wells and/or openings ofthe intermediate substrate are configured to have an area of 0.5 mm² to20 mm², such as 2 mm² to 10 mm², or 2 mm² to 5 mm². In particulardisclosed embodiments, the wells and/or openings of the intermediatesubstrate are circular and have a diameter of 2 mm to 35 mm, such as 6mm to 30 mm, 11 to 22 mm. Representative embodiments comprise wellsand/or openings in the intermediate substrate having a diameter of 2.7mm, 6.4 mm, 6.8 mm, 11 mm, 15.6 mm, 22.1 mm, and 34.8 mm. In someembodiments, the wells and/or openings of the top substrate areconfigured to have an area of 1 mm² to 900 mm², such as 1 mm² to 876mm², or 20 mm² to 340 mm². Representative embodiments comprise wellsand/or openings having an area of 1.33 mm², 19.63 mm², 72.38 mm², 158.37mm², 336.53 mm², and 878.16 mm². In particular disclosed embodiments,the wells and/or openings of the top substrate are circular and have adiameter of 1 mm to 34 mm, such as 1.3 mm to 33.4 mm, or 5 mm to 20 mm.Representative embodiments comprise wells and/or openings in the topsubstrate having a diameter of 1.3 mm, 5 mm, 5.4 mm, 9.6 mm, 14.20 mm,20.7 mm, and 33.4 mm. In some embodiments, the shape and size of eachwell and/or opening of the intermediate and/or top substrates can bevaried such that each well in a plurality of wells and/or openings canhave different sizes and/or different shapes. Solely by way of example,some embodiments can utilize intermediate and/or top substrates thathave gradually increasing or decreasing well and/or opening sizes, suchthat the wells and/or openings in a row of wells and/or openingsincrease in size from left to right (or vice versa).

In some embodiments, the substrate-based analytical devices can furthercomprise a sensor substrate. The sensor substrate can be used to providethe analyte to be evaluated using the substrate-based analytical device,or it can be used to provide one or more signaling agents that interactwith an external sample comprising an analyte. The analyte or signalingagent can be provided with the sensor substrate in a constructed deviceready for use or it can be added to the sensor substrate prior to use.In some embodiments, the sensor substrate can be positioned between thetop paper substrate and the intermediate paper substrate. In someembodiments, the sensor substrate can comprise a substrate configured toinclude a plurality of wells onto which an analyte can be deposited. Insome embodiments, the sensor substrate can be a sheet comprisingtextured areas arranged in a configuration that matches that of thewells and/or openings of the intermediate substrate to which it iscoupled. In some embodiments, a glass fiber material can be used toprovide the textured areas. In yet additional embodiments, the sensorsubstrate can be provided as a plurality of individual substrates uponwhich an analyte can be deposited, such as a plurality of individualglass microfiber substrates. The individual substrates typically arefabricated to have a shape and size matching that of the wells and/oropenings of the intermediate substrate. In such embodiments, theindividual substrates are aligned with the wells and/or openings of theintermediate substrate. The sensor substrate can comprise any materialsuitable for absorbing an analyte. Analytes can be provided neat or as asolution. In particular disclosed embodiments, the sensor substrate ismade of a microfiber material, such as a glass microfiber material or apolymer-filled glass microfiber material; a cellulosic material, such aspaper or nitrocellulose membranes; a fine woven material, such as nylonor other polymers; and natural materials, such as cotton or wool. Inparticular disclosed embodiments, the sensor substrate comprises aborosilicate glass material. In some embodiments, the particular choiceof material can depend on assay conditions, such as reagent composition(e.g., solvent compatibility), and sample composition. Materials may beselected based on the detection mechanism. For example, with solidopaque materials, the ability to read through the material (such as inUV-VIS instruments, which use a top or bottom read with illuminationfrom the opposite side) is reduced and can thereby decrease theobservable signal. This can be avoided by selecting a particularmaterial that improves readability of the signal. In some embodimentsusing fluorescence detection, illumination and detection occur from thesame side and thus one can use a variety of materials so long as thebackground fluorescence is low enough to not interfere with the assay.

A representative strip-based device is illustrated in FIG. 1. Asillustrated in FIG. 1, strip-based device 100 can comprise fourdifferent substrates, such as bottom substrate 102, intermediatesubstrate 104, sensor substrate 106, and top substrate 108. In theembodiment illustrated in FIG. 1, the sensor substrate comprises aplurality of individual sensor substrate substrates 110 that arefabricated to match the size, shape, and pattern of openings 112 of theintermediate substrate 104. Top substrate 108 comprises openings 114,which are fabricated to have a smaller diameter than that of theindividual sensor substrate substrates 110. While a particular number ofopenings (e.g., 12 openings) and individual sensor substrate substrates(e.g., 12 openings) are depicted in FIG. 1, the present disclosure isnot limited to this particular embodiment and contemplates otherembodiments wherein more or fewer individual sensor substrate substratesand/or openings (or wells) are provided. Also, the shape and size ofeach well and/or opening of the intermediate substrate and the topsubstrate can vary as described above.

A representative paper-based well plate embodiment 200 is illustrated inFIG. 2. Paper-based well plate device 200 comprises four substrates, abottom substrate 202, an intermediate substrate 204, a sensor substrate206, and a top substrate 208. Each of the intermediate substrate 204 andthe top substrate 208 is configured to comprise a plurality of rows ofopenings 210 and 212, respectively, to thereby provide a platformrepresenting a well plate device used in various applications, such asanalyte analysis, biological assays, chemical assays, and the like. Thesensor substrate 206 comprises a plurality of individual sensorsubstrate substrates 214 that are fabricated to match the size, shape,and configuration of openings 210 of the intermediate substrate 204. Topsubstrate 208 comprises openings 212, which are fabricated to have asmaller diameter than that of the individual sensor substrate substrates214. While a particular number of openings (e.g., 96 openings) andindividual sensor substrate substrates (e.g., 96 openings) are depictedin FIG. 2, the present disclosure is not limited to this particularembodiment and contemplates other embodiments wherein more or fewerindividual sensor substrate substrates and wells are provided. Also, theshape and size of each opening of the intermediate substrate and the topsubstrate can vary as described above and in some embodiments can bewells rather than openings. Additional representative paper-based wellplate devices are illustrated in FIGS. 3 and 4. In some embodiments, thedevice can be a 6-well plate device, a 24-well plate device, 36-wellplate device, a 96-well plate device, a 384-well plate device, a1080-well plate device, a 1536-well plate device, and so on.

As indicated above, the devices described herein can be modified tocomprise a signaling agent. For example, devices can be fabricated witha sensor substrate in which a signaling agent is embedded. Suitablesignaling agents can include, but are not limited to, fluorescentcompounds (e.g., fluorophores), chromogenic compounds (e.g., dyes),quantum dots, a member of a specific binding pair (which can emit asignal upon specific binding with another member of the specific bindingpair), and the like. In some embodiments, the substrate-based analyticaldevices can further comprise a separate signaling agent substratecomprising wells in which a signaling agent can be deposited. Thesignaling agent substrate can be positioned within the substrate-basedanalytical device such that it is placed between an intermediatesubstrate of the device and a sensor substrate of the device, or suchthat it is placed between a top substrate of the device and a sensorsubstrate of the device. In additional embodiments, the devices can bemodified with chemical reagents typically used in clinical analyticalmethods. The chemical reagents can be provided by the sensor substrateor another substrate added into the device.

The devices disclosed herein also can comprise one or more stackinglayers that are introduced to increase the volume of sample that thedevice can hold and/or to provide multiple different reagents foranalysis. In some embodiments, a plurality of stacking layers havinghydrophilic sections and/or openings configured to match the shape,size, and/or pattern of wells and/or openings of a top and/orintermediate substrate can be provided. These hydrophilic sectionsprovide the ability to absorb more sample when using the device as thereare multiple layers of hydrophilic surfaces into which the sample can beabsorbed. In yet additional embodiments, multiple stacking layers can beused wherein each stacking layer has regions in which a differentreagent has been absorbed, these regions matching the shape, size,and/or pattern of wells and/or openings of the top and/or intermediatesubstrates. As such, when a sample is introduced into the wells and/oropenings of the device, it can pass through multiple different reagentlayers provided by the stacking layers. In such embodiments, atransparent bottom layer can be used so that any generated signal can bedetected (e.g., visually or using a spectroscopic technique, such asUV-Vis spectroscopy) after the sample has passed through the differentlayers of the device. Exemplary transparent bottom layers can be madefrom materials such as polycarbonates, polystyrenes, and PET.Alternatively, a non-transparent or opaque bottom layer may be used suchthat detection can occur from viewing the top of the device. Anexemplary stacking layer 502 is illustrated in device 500 illustrated inFIG. 5, which also comprises an intermediate substrate 504, a pluralityof sensor substrates 506, and a top substrate 508.

Also disclosed herein are embodiments of hybrid microfluidicpolymer-based analytical devices. Such devices integrate fluidicchannels in combination with the well plate or strip device embodimentsdescribed herein. For example, a microfluidic architecture of channelscan be embedded in a substrate such that the different wells and/oropenings of a substrate can be connected via the channels. In thesedevices, the channels are defined in a manner equivalent to that whichdefines the wells and/or openings, by use of a hydrophobic barrier. Thechannels can be low-volume flow paths that connect the wells and/oropenings while ensuring that only a minimal volume of the sample or asolvent may be retained in the channels following their use. See, forexample, FIGS. 6, 7, and 8, which illustrate well plate embodimentswherein multiple wells and/or openings are connected with fluidicchannels. As illustrated in FIG. 6, device 600 comprises plural sets ofthree openings 602, which are made in the intermediate substrate 604 andare connected via fluidic channel 606. One opening (e.g., 608) can serveas a sample introduction zone; a second fluidly coupled opening (e.g.,610) can serve as a reagent zone; and a third fluidly coupled opening(e.g., 612) can serve as a detection zone. Device 600 further comprisesa stacking substrate 614, which can be made from opaque or transparentmaterials; a plurality of sensor substrates 616; and a top substrate618. In yet additional embodiments, a stacking layer can be configuredto act as a sump that can work with a one-way valve that is used tocontrol flow through fluidic channels that connect different wellsand/or openings of a substrate of the device. In some embodiments, thevalve can comprise a low melting polymer (e.g., PCL or anotherhydrophobic polymer disclosed herein), solid oils (e.g., coconut oil,hydrogenated coconut oil, and the like), and/or wax (e.g., paraffin wax,beeswax, and the like) that can be melted using a suitable technique(e.g., a CO₂ laser). The valves can be actuated (that is, opened) byselectively melting the low melting polymer valve, such as by focusing alow-energy laser beam at the desired region to be melted. In yetadditional embodiments, a metallic nanoparticle ink can beinkjet-printed onto the back of a given layer (e.g., bottom layer orintermediate layer) to build a heater element. Such embodiments can beused to deliver a given temperature for purposes of valve actuation(e.g., having heater elements both above and below could allow for thevalves to be more than just single-use), or to provide the capability toincubate a reaction or other process. The materials making up the valvescan be selected to have different melting points, but it also isfeasible to have a single base material in two molecular weightincrements having differing melting points. The low melting polymerlayer positioned above the substrate comprising the actuated valve canbe selectively heated to facilitate melting of the low melting polymerlayer such that the low melting polymer is able to refill the spacepreviously occupied by the valve prior to actuation. By allowing the lowmelting polymer to cool, the valve can be reformed because the lowmelting polymer will solidify within the fluidic channel. In yet someembodiments, a two-stage valve system can be provided by using two lowmelting components, wherein one melts at a lower temperature than theother so that a first valve can be operated and then the second valveprovided by the second higher melting component can be actuated using ahigher melting temperature. For example, a two-stage valve system couldinvolve opening the first valve at 24° C. and then further increasingthe temp to 36° C. (or higher) to open the second valve using a built-inincubation component and or a laser or other method of localized heating(e.g., custom electrically-driven heaters positioned in an area near thevalve material). A built-in heater could comprise adding an etched foilsilicon-rubber/polyester heater circuit as an intermediate layer of thedevice or by inserting a NiChrome wire in a layer of the device. Bymelting the polymer or wax, the channel is opened so that fluid can flowthrough the fluidic channel into a fluidly connected well or opening. Insome embodiments, a layer of low melting polymer and/or wax can be addedbetween substrates of the device to provide a mechanism for reclosingsuch valves. Solely by way of example, a layer of a low melting polymercan be included above a substrate comprising fluidic channels withvalves as discussed above.

A representative hybrid well plate device is illustrated in FIGS. 7 and8. As illustrated in FIG. 7, different wells and/or openings can befluidly connected using fluidic channels. According to device embodiment700 of FIG. 7, an intermediate substrate 702 is configured to comprise aplurality of fluidic channels 704 embedded within surface 706 ofsubstrate 702. Multiple different fluidic channel configurations can beused. For example, fluidic channels 704 a and 704 b are used to providea linear configuration between openings 708, 710, and 712. In anotherexample, fluidic channels 704 c and 704 d can provide a “v-shaped”configuration between openings 714, 716, and 718. Other exemplaryfluidic channel configurations are illustrated in FIG. 7. Also, asillustrated in FIG. 7, multiple different sensor substrates 720 can beprovided. These sensor substrates can be arranged in configurations thatmatch different opening/fluidic channel patterns provided inintermediate substrate 702. For example, sensor substrates 720 a, 720 b,and 720 c can be arranged in a linear configuration and connected via ahydrophilic portion 722 which is configured to fit within fluidicchannels 704 a and 704 b. A top substrate 724 also is provided which hasa plurality of openings 726 that allow one to view any signals generatedwithin one or more openings of the intermediate substrate.

The substrates of the disclosed substrate-based analytical devices canhave thicknesses that are the same or different from one another. Insome embodiments, the bottom and intermediate substrates can havethicknesses ranging from 0.45 mm to 1.35 mm, such as 0.45 mm to 0.9 mm,or 0.5 mm to 0.8 mm. In representative embodiments, the bottom andintermediate substrates are 0.45 mm thick. In some embodiments, the topsubstrate can have a thickness ranging from 0.1 mm to 0.45 mm, such as0.1 mm to 0.2 mm, or 0.1 mm to 0.15 mm. In representative embodiments,the top substrate is 0.15 mm thick. In some embodiments, the stackinglayers and/or sensor layers described above can have similar thicknessesas the bottom, top, and/or intermediate substrates. In particulardisclosed embodiments, the sensor layer is thinner than the other layersto provide tighter connections between the different layers.

The hydrophobic polymer component of the disclosed substrate-basedanalytical devices can be a polymer having a structure satisfying aFormula I

With reference to Formula 1, Z, Y, and W independently can be O, S, NH,or NR², where R² is selected from hydrogen, aliphatic, aryl, andheteroaryl; each of R³, R⁴, R⁵ and R⁶ (if present) independently can behydrogen, aliphatic, aryl, heteroaryl, or a heteroatom-containing moiety(e.g., halogen; aldehyde (—R^(a)CHO); acyl halide (—R^(a)C(O)X, where Xis selected from fluorine, chlorine, bromine, and iodine); carbonate(—R^(a)OC(O)OR^(b)); carboxyl (—R^(a)C(O)OH); carboxylate (—R^(a)COO⁻);ether (—R^(a)OR^(b)); ester (—R^(a)C(O)OR^(b), or —R^(a)OC(O)R^(b));hydroxyl (—R^(a)OH); ketone (—R^(a)C(O)R^(b)); silyl ether(R^(b)R^(c)R^(d)SiOR^(a)—); peroxy (—R^(a)OOR^(b)); hydroperoxy(—R^(a)OOH); phosphate (—R^(a)OP(O)(OH)₂); phosphoryl (—R^(a)P(O)(OH)₂);phosphine (—PR^(a)R^(b)R^(c)); thiol (—R^(a)SH); thioether/sulfide(—R^(a)SR); disulfide (—R^(a)SSR^(b)); sulfinyl (—R^(a)S(O)R^(b));sulfonyl (—R^(a)SO₂R^(b)); carbonothioyl (—R^(a)C(S)R^(b) or—R^(a)C(S)H); sulfino (—R^(a)S(O)OH); sulfo (—R^(a)SO₃H); thiocyanate(—R^(a)SCN); isothiocyanate (—R^(a)NCS); oxazole; oxadiazole; imidazole;triazole; tetrazole; amide (—R^(a)C(O)NR^(b)R^(c), or—R^(a)NR^(b)C(O)R^(c)); azide (N₃); azo (—R^(a)NNR^(b)); cyano(—R^(a)SCN); isocyanate (—R^(a)NCO); imide (—R^(a)C(O)NR^(b)C(O)R^(c));nitrile (—R^(a)CN); isonitrile (—R^(a)N⁺C⁻); nitro (—R^(a)NO₂); nitroso(—R^(a)NO); nitromethyl (—R^(a)CH₂NO₂); and amine (—R^(a)NH₂,—R^(a)NHR^(b), —R^(a)NR^(b)R^(c)); wherein R^(a) is a bond, aliphatic,aryl, heteroaliphatic, or heteroaryl; R^(b), R^(c), and R^(d)independently are hydrogen, aliphatic, aryl, heteroaliphatic,heteroaryl, or any combination thereof; r is an integer selected from 1to 4; s and t independently are integers selected from 0 to 4; and q isan integer selected from 1 to 1000.

In particular disclosed embodiments, the hydrophobic polymer componenthas a structure satisfying Formula II or Formula III:

In particular disclosed embodiments, the hydrophobic polymer has astructure satisfying Formula IV.

In yet additional embodiments, the polymer can be a polyvinyl polymer,such as polyethylene, polypropylene, polyvinyl chloride, or combinationsthereof. Certain embodiments concern using polycaprolactone (alsoreferred to herein as “PCL”), polycaprolactone diol, polycaprolactonetriol, polycaprolactone-block-polytetrahydrofuan-block polycaprolactone,poly(ethylene oxide)-block-polycaprolactone, poly(ethyleneglycol)-block-poly(e-caprolactone) methyl ether, polyvinyl chloride, orany combinations thereof as the hydrophobic polymer.

The hydrophobic polymer typically is used to coat the substrates of thedevice as described herein. In particular disclosed embodiments, acoating of the hydrophobic polymer can have a thickness ranging fromsurface films of 0.01 μm to 5 μm or may consist of an embedded(permeating) format. In such permeating embodiments, a particular amountof the hydrophobic polymer can be added to the substrates such that itpartially permeates or completely saturates the substrates. In suchembodiments, the amount of the polymer added can range from 5 mL to 15mL of 5% PCL in toluene, such as 8 mL to 10 mL of 5% PCL in toluene, or10 mL of 5% PCL in toluene. In particular embodiments the hydrophobicpolymer is entirely within the matrix of the support material from adepth of 0.1% to 100% of the depth of the substrate.

Also disclosed herein are embodiments of additional device componentsthat can be used in combination with the substrate-based analyticaldevices disclosed herein. For example, a plate frame, an alignmentcomponent, and a combined device comprising these components aredisclosed. The plate frame and the alignment component can be used tohouse a strip-based device or a well plate device as described abovesuch that proper alignment of the components of the disclosedstrip-based and well plate devices is obtained and that thesubstrate-based analytical devices further are properly aligned withplate readers and/or liquid handling instrumentation that may be used inanalytical techniques using the disclosed devices. In some embodiments,the plate frame is a well plate frame that is fabricated to hold a6-well plate device, a 24-well plate device, 36-well plate device, a96-well plate device, a 384-well plate device, a 1080-well plate device,or a 1536-well plate device described herein.

In particular disclosed embodiments, the plate frame and alignmentcomponent are reusable and can be fabricated using 3-dimensionalprinting. The plate frame component typically comprises first and secondcomponents. In some embodiments, the second component can be fabricatedto match (or substantially match) the dimensions of the substrate-basedstrip and/or substrate-based well plate device to be used with theframe. In some embodiments, the first component can facilitate clampingthe substrate-based analytical device in place and can be fabricated tocomprise a plurality of openings that match a configuration of wellsand/or openings in a substrate-based device described herein. Otherconfigurations of the first and second components are disclosed herein.In some embodiments, an alignment component can be fabricated that has ashape similar to the second component of the plate frame and that fitsaround the exterior of the second component to facilitate alignment asdiscussed above. The plate frame and the alignment device can bedesigned to have any desired measurements using a suitable softwareprogram and then can be printed using a biodegradable polymeric material(e.g., polylactic acid (PLA)) and a 3-dimensional printer. In someembodiments, the components of the plate frame each comprise an outerperimeter section that is configured to align with the outer perimeterof the other component or to fit within or over the outer perimeter ofthe other component. When the first and second components are associatedtogether, they will define a chamber that is capable of holding in placeany one or more of the polymer-based analytical devices describedherein. These components can facilitate use of the disclosedsubstrate-based analytical devices with benchtop components used invarious analysis-based technologies, such as plate readers, platewashers, plate stackers/handlers, and dispensing components.

In some embodiments, magnets (e.g., Neodymium magnets) can be embeddedin the first and second components of the plate frame (e.g., six magnetsper section) at positions that will become aligned when the first andsecond components are pieced together (e.g., in the outer perimetersections of the first and/or second components), thus allowing forstrong clamping and self-alignment of the frame pieces around thesubstrate-based analytical devices. The magnets also can facilitatereplacement of the substrate-based device while allowing the plate frameto be reused, thus reducing waste associated with well plate basedassays.

A representative embodiment of a plate frame is illustrated in FIG. 9.Plate frame 900 comprises a second component 902 (which acts as thebottom of the plate frame) and a first component 904 (which acts as thetop of the plate frame). First component 904 comprises housings 906 thatare configured house magnets and that are configured to match thelocations of housings 908, which also can house magnets, provided insecond component 902. Another embodiment of a plate frame is illustratedin FIG. 10. FIG. 10 is an exploded perspective view of a plate framedevice 1000, which comprises a second component 1002 and a firstcomponent 1004. As illustrated in FIG. 10, a plurality of openings 1006can be made in the first component 1004, which are configured to matchthe configurations of the openings and/or wells of the well plate deviceplaced in the holder such that a user can visualize signals generatedusing the well plate device. Second component comprises walls 1008 and1010 and first component 1004 comprises walls 1012 and 1014. In theembodiment illustrated in FIG. 10, the first component 1004 and secondcomponent 1002 can be fit together such that walls 1008 and 1010 of thesecond component fit within walls 1012 and 1014 of first component 1004.In some embodiments, device 1000 can be modified to include magnets inone or more of walls 1008, 1010, 1012, and 1014 so that magnets in wallsof the first component are magnetically attracted to magnets withinwalls of the second component. In some embodiments, the magnets can beembedded within walls 1008, 1010, 1012, and 1014 or they can be added tothe exterior and/or interior of these walls.

FIGS. 11A and 11B are photographic images of representative first andsecond components of a plate frame device. FIG. 12 is an illustration ofan alignment component that can be used to align the first and secondcomponents when combined with different substrates described herein.FIGS. 13A-13D show representative plate frame and alignment componentsat various stages of implementation. FIG. 13A shows an alignmentcomponent; FIG. 13B shows the alignment component combined with a secondcomponent of a plate frame; FIG. 13C shows the positioning of a wellplate device within the plate frame and alignment component and FIG. 13Dshows the well plate device within the completed plate frame after thealignment component is removed.

IV. Methods of Making Devices

Disclosed herein are embodiments of methods for making thesubstrate-based analytical devices described herein.

In some embodiments, the methods can comprise depositing a hydrophobicpolymer as described herein on a substrate of the device (e.g., a bottomsubstrate, an intermediate substrate, a top substrate, or anycombination thereof). In some embodiments, the hydrophobic polymer canbe deposited as a solution (e.g., wherein the hydrophobic polymer isdissolved in a solvent, such as toluene) or as a thin film (e.g.,wherein the hydrophobic polymer is melted and then deposited as a thinfilm without using a solvent). Using the hydrophobic polymer, thechemical properties of the substrate can be modified such thathydrophilic substrates are converted to hydrophobic substrates (e.g., bycoating the substrate or a portion thereof with the hydrophobicpolymer). In yet additional embodiments, non-hydrophilic substrates canbe rendered hydrophilic using the hydrophobic polymer. For example, anon-hydrophilic substrate can be coated with the hydrophobic polymer andthen a portion of the substrate comprising the hydrophobic polymer canbe treated with O₂ plasma to render the treated surface hydrophilic. Insome embodiments the substrate itself can be treated with an O₂ plasmatreatment to render the substrate surface hydrophilic. Hydrophilicregions formed on a substrate by treating only a portion of thesubstrate (or by treating a masked substrate as described below) withthe hydrophobic polymer serve as wells of the substrate in someembodiments.

The methods can further comprise masking a substrate of the device, suchas an intermediate substrate or a top substrate, with a suitable maskingagent (e.g., a masking tape) prior to depositing the hydrophobicpolymer. The masked substrate can then be patterned using a suitablepatterning device, such as a laser cutter, plotting cutter, or evenmanual cutting. After patterning, a portion of the masking agent can beremoved from the substrate and the substrate can be covered with asolution of the hydrophobic polymer using any suitable technique (e.g.,airbrushing, spraying, dipping, inkjet deposition, or the like), therebyrendering the unmasked regions of the substrate hydrophobic. Thesubstrate can then be dried (using an affirmative drying step where thesubstrate is exposed to heat, air flow, or an inert gas flow; or simplyallowing the substrate to dry in ambient atmosphere). The remainingmasking agent can then be removed to expose a patterned substratecomprising hydrophilic regions. In yet additional embodiments,substrates that are to be patterned can first be coated with a solutionof the hydrophobic polymer and then patterned by cutting the desiredpattern into the polymer-coated substrate (using cutting techniquesdescribed above). Patterned regions of the substrate can then be treatedwith an O₂ plasma treatment to render the patterned regions hydrophilicthereby forming wells. In some embodiments, fluidic channels can beformed in the substrates to join different wells and/or openings. Forexample, fluidic channels can be laser (or manually) cut into thehydrophobic polymer component surrounding the perimeters of the wellsand/or openings and thus expose a region of the substrate which canserve as a channel through which fluid can flow from one well/opening toanother. In yet some additional embodiments, well and/or channelarchitectures can be patterned on a substrate (e.g., an intermediatesubstrate) by screen printing a polymer onto the substrate to therebydefine hydrophilic wells and/or channels. In some embodiments, screenprinting facilitates the ability to produce substrates having differentarchitectures, such as single wells, or wells that are connected througha fluidic channel. Such connections can facilitate fluid transfer fromone well to another. For example, as illustrated in FIGS. 14A-14C, thepatterned substrate 1400 (shown in FIG. 14A), which comprises threedifferent well/fluidic channel configurations 1402, can be used in adevice to facilitate transferring assay components from one well 1404 toanother well 1404 via a fluidic channel 1406. The device shown in FIG.14B comprises a top substrate 1408 configured with openings that allowthe use to visualize the wells of patterned substrate 1400. The deviceshown in FIG. 14C includes a top substrate 1410 that is configured tocomprise wells and fluidic channels matching those of substrate 1400 sothat fluid movement through the fluidic channels can be visualized. Asfurther illustrated by FIGS. 15A-15C, well and fluidic channelconfigurations like those illustrated in FIGS. 14A-14C can be used totransfer reagents and/or assay components from well to well. Differentlydyed fluids are used in FIGS. 15A and 15C to illustrate fluid flowthrough the device. As shown by FIG. 15B, an assay component(represented by blue dye) is immobilized on wells positioned in columnb, rows 2 and 3. The previously deposited assay component isreconstituted with the sample flow (represented by red dye). Once thesample is added to the well of column a, row 3, the previously depositedassay component on the well of column b, row 3 re-dissolves and themixture (containing both red and blue dyes) will travel to the well ofcolumn c, row 3.

The fluidic channels of device embodiments described herein can have anysuitable dimension, such as 25 μm to 5 mm (or higher) with the averagewidth in the range of 100 μm to 250 μm. The height of the channeltypically is defined by the thickness of the layer. For example, ifstandard filter paper is used, the channel could have a thicknessranging from 150 μm to 200 μm. In some embodiments, the length of thechannel can be on the order of several millimeters in length. Forexample, lower density plates (e.g., 96-well plates) can have channelson the order of several mm, while higher density plates (e.g., >384-wellplates) may have much shorter channels (e.g., micrometer and/ornanometer length channels).

For substrates that do not require patterning (e.g., a bottomsubstrate), the substrate can simply be coated with a solution of thehydrophobic polymer without patterning. These substrates can be driedand then combined with the other substrates of the device using a manualcoupling technique (e.g., stacking the substrates and then holding themtogether with a clamping mechanism) or using a laminating couplingtechnique (e.g., stacking the substrates together and then using athermal laminator to press the substrates together). For example, insome embodiments, a top substrate, a sensor substrate, and anintermediate substrate can be stacked such that the sensor substrate ispositioned between the top and intermediate substrates. These substratescan then be laminated together. Then a bottom substrate can be laminatedto these combined substrates. In yet additional embodiments, multiplesubstrates with openings, wells, and/or channels can be stacked suchthat a device comprises one or more substrates with openings, one ormore substrates with wells, one or more non-patterned substrates, andany combinations thereof. An exemplary stacked device 1600 isillustrated in FIG. 16A. An exploded perspective view of device 1600 isillustrated in FIG. 16B. As illustrated in FIG. 16B, a colored (orotherwise non-transparent) top substrate 1602 comprising a plurality ofopenings 1604 is fluidly coupled with a first intermediate substrate1606, which also is configured to comprise a plurality of openings 1608.This first intermediate substrate 1606 also is fluidly coupled with asecond intermediate substrate 1610, which is configured to comprise aplurality of wells 1612 in a pattern matching openings 1604 and 1608 oftop substrate 1602 and first intermediate substrate 1606. Non-patternedsubstrate 1614, which is coated with a hydrophobic polymer is associatedwith second intermediate substrate 1610 and also bottom, coloredsubstrate 1616. All of these components are stacked and coupled togetherto provide constructed device 1600 as illustrated in FIG. 16A.

In additional embodiments, individual substrates (e.g., bottom,intermediate, and top substrates) of the device can be saturated with asolution of the hydrophobic polymer. The substrates can then be driedusing techniques described above to provide an even distribution of thepolymer on all surfaces of the substrate(s). A desired pattern can thenbe cut into one or more substrates (such as to provide an intermediateand/or top substrate). Patterned and non-patterned substrates are thenstacked in a configuration described herein and laminated at atemperature suitable to melt the hydrophobic polymer (e.g., temperaturesranging from 60° C. to 120° C., such as 90° C. to 110° C., or 100° C. to110° C.).

In yet additional embodiments, the methods described herein can compriseapplying an even layer of the hydrophobic polymer (either as a solutionor by adhering a film of the hydrophobic polymer) on a surface of asubstrate, such as a polymeric sheet (e.g., a PET-based high temperaturecopier transparency sheet). The even layer of the hydrophobic polymercan be applied using a suitable coating technique, such as airbrushing,spraying, dipping, inkjet deposition, or the like. The substrate is thendried using techniques described above and then optionally patterned(such as to prepare an intermediate or top substrate). A bottomsubstrate, intermediate substrate, and top substrate made according tothis method can then be combined with a sensor substrate such that theintermediate substrate is stacked on top of the bottom substrate,followed by the sensor substrate, and then the top substrate. In someembodiments, the polymeric sheet can be treated for use in fluorescencetechniques by coating the polymeric sheet with a colored ink solution orwith a layer of colored paper.

In embodiments described herein, the hydrophobic polymer not only can beused to coat the different substrates of the disclosed substrate-basedanalytical devices and/or to provide a defined pattern within or on asubstrate of the device, but it also serves as a bonding agent to securethe different substrates together.

V. Methods of Use

In some embodiments, the disclosed substrate-based analytical devicesand additional device components can be used for analytical analysis andhigh-throughput screening and analysis. In particular disclosedembodiments, the devices disclosed herein can be used for analytedetection techniques, such as detecting environmental or health toxins,chemical compounds (e.g., cyanide or other water contaminants), diseasebiomarkers, and the like. In additional embodiments, the devices can beused to complete multi-step assays that are typically conducted in wellplates and/or multi-step assays that require multiple reagent additionsand incubations, which can be conducted in a single step using thedisclosed devices. For example, see FIGS. 6, 7, and 8, which illustratedevice embodiments that can be used for plural assays at the same time.Device embodiments comprising polymeric substrates can be used for topor bottom reading in colorimetric and fluorescent/luminescent assays. Inyet additional embodiments, the devices can be pre-fabricated withreagents used for clinical chemistry panels used in the medical field.

In some embodiments, the devices can comprise (or can be modified tocomprise) a sensor substrate comprising a signaling agent that, oncecontacted with an analyte, can emit a signal, such as a fluorescentsignal, colorimetric signal, or other signals described herein. Inparticular disclosed embodiments, the sensor substrate comprising thesignaling agent can be exposed to an external sample (e.g., biologicalsample obtained from a subject or an environmental sample, such aswater, air, soil, plants, or the like) in order to determine if theexternal sample comprises an analyte of interest. In other embodiments,the sample can be provided as part of the sensor substrate and thesignaling agent can be added to the device so as to determine if thesample comprises an analyte of interest. In some embodiments, thedevices can be used to quantify the amount of analyte present in thesample. For example, if a signal is emitted upon exposure of thesignaling agent to an analyte, then this can correspond to a particularconcentration of the analyte. In yet additional embodiments, the devicescan be used to qualify the analyte present in the sample. For example,the generation of a signal (e.g., a particular color change,fluorescence, or quenched fluorescence) can indicate that a particularanalyte is present in the sample. In some embodiments, the device can beused in combination with a reference chart (e.g., a color chart) thatprovides a reference for quantifying and/or qualifying the analytedetected using the disclosed devices. For example, a color chart can beused as a reference to determine a particular type of analyte present ina sample based on the color signal emitted from the sensor substrateafter using the device. A user can visually compare the color signalemitted from the sensor substrate and can match it to a color on thecolor chart that correlates a particular type of analyte. In yetadditional embodiments, the devices can be used in combination with auser's pre-existing laboratory plate reader for quantitative and/orqualitative analysis.

Embodiments utilizing fluidic channels connecting differentwells/openings of a patterned substrate allow for complex analyticmethods to be conducted using the disclosed substrate-based devices. Forexample, by connecting multiple wells/openings with fluidic channels,sequential steps of an assay can be performed, or multiple tests of asingle sample can be conducted. Solely by way of example, a plurality ofwells/openings can be joined with fluidic channels to provide (i) asample introduction zone, which can be configured to accept a sample ora sensor substrate comprising a sample, (ii) a reagent zone, which canbe configured to accept a sensor substrate comprising particularreagents for the type of test being conducted, (iii) and a detectionzone, which can be configured to accept a sensor substrate comprising asignal generating moiety or a sensor substrate that emits an observableor measurable signal upon exposure to a fluid delivered from the reagentzone. Channels with fluidic channels can be used in techniques, such asabsorbance spectroscopy (through the short or long dimension of thechannel), emission spectroscopy, extraction, chromatography,electrophoresis, affinity sorptive processes, molecular recognitionprocesses, electrochemical measurement processes, solution conductanceor impedance measurements, and interfacing to external systems includingspectrometers and other process or detection platforms.

In some embodiments, the disclosed devices are used for cyanidedetection as illustrated schematically in FIG. 17. Methods using thedisclosed devices for cyanide detection are discussed below. The methodsdescribed below can be adapted for detection of other analytes capableof displacing ions (e.g., copper ions) from the surface of a quantumdot. In representative embodiments, a paper-based well plate device isuse in combination with chitosan encapsulated CdTe quantum dots, havinga maximum emission of 520 nm (CS-QD520), as fluorophores. Such quantumdots are specifically quenched by copper (II) ions. The quenchedchitosan-QD nanoparticle are used as a sensor probe for cyanidedetection. Upon cyanide introduction, the fluorescent signal isrecovered due to the formation of copper cyanide complex[Cu(CN)_(x)]^(1-x), thus freeing the chitosan-QD nanoparticle. The“signal-ON” fluorescence is graphed against cyanide at clinical relevantconcentrations. The fluorescent assay can be pre-concentrated on thesurface of a glass microfiber filter, which serves as a sensor substrateof the device. Such embodiments provide a higher signal compared tosolution-based assays due to the higher surface-to-volume ratio of theassay. Each well (or opening) of the well plate can be run as anindependent assay, which is able to analyze multiple samplessimultaneously.

VI. Examples

Reagents—All the chemicals used were of analytical-reagent grade.Deionized water was obtained from a Milli-Q® Advantage A10. CdCl₂(anhydrous, 99%), tellurium powder (˜200 mesh, 99.8%),3-mercaptopropionic acid (MPA, 99%), Sodium borohydride (NaBH₄, 98%),Chitosan (MW=50-190 KDa, 75-85% deacetylated) were purchased fromSigma-Aldrich (St. Louis, Mo., USA). Polycaprolactone (PCL, Capa™ 6800,MW=80 000) was obtained from Perstorp (Cheshire UK). Copper sulfate(CuSO₄.5H₂O) was purchased from Fisher Chemical (Fair Lawn, N.J., USA).Potassium Cyanide (KCN) was obtained from Mallinckrodt (Phillipsburg,N.J., USA). Black filter paper was purchased from Ahlstrom FiltrationLLC (Mt. Holly Springs, Pa., USA). GF/B microfiber filter was purchasedfrom Whatman GE healthcare (Buckinghamshire, UK). Black poster paperboard was purchased from UCreate poster board, 22″×28″ (Charlotte, N.C.,USA).

Synthesis of Core CdTe Quantum Dot

CdTe Quantum dots were synthesized in aqueous solution as previouslydescribed with some modifications. Two mmol anhydrous CdCl₂ (0.3666 g)and 4.8 mmol 3-mercaptopropionic acid (418 μL) were mixed in 200 mL ofMilli-Q water. The pH of the solution was adjusted to 11 using 1.0 MNaOH. The mixture was purged with N₂ gas for 30 min to eliminate oxygenfrom solution. The NaHTe solution was prepared separately by mixing 0.5mmol tellurium powder (63.8 mg) and 5 mmol NaBH₄ (0.1891 g) in 5 mLMilli-Q water. The NaHTe precursor was added to the cadmium precursorsolution and the mixture was refluxed at 95-100° C. for 3 hr. The finalmolar ratio of Cd²⁺/MPA/NaHTe was 1:2.4:0.25, respectively. The averagediameter and concentration of CdTe QDs were calculated as 1.76 nm and81.7 μM, respectively.

Synthesis of Chitosan Encapsulated CdTe Quantum Dot (CS-QD)

Chitosan stock solution (1% w/v) was prepared by dissolving 1 g chitosanin 100 mL of 1% acetic acid. The stock solution was subsequently dilutedto 0.01% by 0.1% acetic acid. As prepared CdTe QD solution (22 mL) wasgradually added in 100 mL of 0.01% chitosan solution with stirringfollowed by addition of 1 mL EDC (10 mg/mL)/NHS (5 mg/mL) solution. Themixture was further stirred overnight at room temperature to formcovalent bonds between carboxylic acid on quantum dot surface and aminogroup on chitosan. The chitosan encapsulated CdTe quantum dot was washed3 times with Milli-Q water by centrifugation. In the final step, theprecipitate was re-dispersed and stored in 22 mL (the same volume asQD520) phosphate buffer 10 mM, pH 7.4. The concentration of CS-QD wouldbe 81.7 μM derived from QD520 concentration. The mass/volumeconcentration is 3.76 mg/mL.

Cyanide Probe—Quenching of CS-QD520 by Copper Ion

The stock chitosan-QD solution was diluted 10 fold with Tris-HCl buffer10 mM pH 7. A certain amount of copper (II) standard (100 mg/L) wasadded to the diluted chitosan-QD solution with stirring to quench thefluorescence signal of CSQD520. The quenching reaction was continued for3 hours. Twenty microliters of the quenched solution (Cu-CSQD520) wasdrop-casted on a circle piece of glass microfiber filters, grade GF/B(6.8 mm diameter) cut by a hole punch. The sensor dot was driedovernight at 40° C. in a vacuum oven.

Example 1

In this example, the paper based strip/plate was designed and drawn toresemble a standard 96-plastic well plate using SolidWorks® 2013(Dassault Systèmes, Waltham, Mass., USA) shown in FIG. 2. The designcontained three layers: bottom, middle, and top. The bottom and middlelayer were composed of black poster paper (0.45 mm thickness). The toplayer was composed of black filter paper (0.15 mm thickness). Eachopening on the middle layer has a diameter of 6.8 mm to match with theGF/B sensor dot. The top layer contained openings with a diameter of 5.4mm, which was smaller than the openings on the middle layer. This helpedlock the GF/B sensor dots in place in the middle layer. The poster paperand the filter paper were cut using LASER cutter. All three layers andGF/B dot sensor were assembled together by air-brushing 5%polycaprolactone (PCL) in toluene in between the layers and the topsurface. This made a hydrophobic barrier around each individual openingin the paper plate. Not only being used as a hydrophobic boundary, thePCL in-between layers also functioned as an adhesive, attaching eachplate layer together.

A low-cost thermal laminator (Scotch, model TL902A) was used to pressall layers together resulting in the melting of PCL on the papersurfaces. Two layers were laminated at a time; staring from Top andMiddle layer, aligning GF/B sensor dots in the openings, and lastlylaminating the bottom layer. PCL enabled hydrophobic barrier betweeneach individual opening for each individual assay on the plate. Thedisposable strip/plate was stored in the vacuum-sealed bag until usage.The paper-based well plate was designed to be placed in a plate holder(described below), which was compatible to a tray of any standard platereader.

Example 2

Disposable paper based strips or plates described above were equippedwith plastic holders and ready for the detection. Twenty microliters ofcyanide standards or samples were applied directly to the paper basedstrip/well plate. The liquid sample absorbed on the GF/B substrate andslowly reacted with the Cu-CSQD520 on the top surface of the glassmembrane filter. A fluorescence signal was measured at 520 nm with theexcitation wavelength at 350 nm.

Chitosan encapsulated CdTe quantum dots with a maximum emission of 520nm were synthesized in an aqueous solution and used as a fluorophore.The CS-QDs nanoparticles formed via the electrostatic attraction betweenpositive charge of amino functional group on chitosan and negativecharge of MPA on the QD surface. Both chitosan and QDs were covalentlylinked through amide bond formation by carbodiimide chemistry. The scanemission spectra of QD520 and CS-QD520 showed in FIG. 18A indicated thatfluorescence intensity of CS-QD520 was 3.5 times higher than those ofthe original QD520 at the same mass/volume concentration. Thisexceptional phenomenon may result from the intrinsic properties ofchitosan. Numerous amino and hydroxyl functional groups on thebiopolymer are good capping agents, which helped stabilized the QDssurface. The morphology of CS-QD520 was studied by TEM (FEI Titan withChemiSTEM mode) as shown in FIG. 18B, which revealed the irregular shapeof the nanoparticles with the particle size about 10-25 nm. Theelemental analysis from ChemiSTEM mode in FIG. 18C confirmed thepresence of cadmium and telluride element in the chitosan encapsulatedCdTe QDs.

Chitosan was incorporated into CdTe QDs to facilitate its use on apaper-based well plate format. Chitosan is a biocompatible andbiodegradable polymer, lending to its use in this example. Due to thefact that chitosan contains plenty of amino groups on the side chain,they are readily available for bioconjugation, exhibiting a positivecharge at acidic to neutral pH (pKa of D-glucosamine unit=6.5-7.0). WhenCS-QD520 was applied on the glass microfiber filter containing silanolgroup, the chitosan-QD nanoparticle electrostatically adsorbed only onthe top of the substrate, while QD520 thoroughly absorbed into thesubstrate. The retention capability of chitosan encapsulated QDs alsowas evaluated as shown in FIGS. 19A and 19B. The CS-QD520 permanentlyretained on the top of the substrate after flushing with 10 mL of PBbuffer. The CS-QD520 remained a tight narrow band on the top of theGF/B, whereas the original QD520 leached off from the substrate. Havinga low fluorescence background and loading volume capacity, the glassmicrofiber filter enabled the retention of CS-QD520 on its top surface.This improved the homogeneity of fluorescence signal, promoting its useon paper-based format.

The performance of the well plate was evaluated for PCL hydrophobicity.Two parameters were assessed, namely volume of liquid sample andretention time for liquid to be held in the openings. These twoparameters related to the degree of contamination from opening toopening. Food coloring was used and represented as a liquid sampleapplied on the plate so that the visual inspection can be determined.With 5% PCL sprayed on the paper, the loading capacity of the liquidsample was 25 μL/opening. When the percentage of PCL was increased to10%, the sample loading capacity raised up to 30 μL.

According to the fabrication design of the well plate as a stackinglayer, the sample loading capacity of the paper-based well plate can beincreased by inserting another layer in the middle for a larger volume.In one example, the loading capacity was increased up to 40 μL with a4-layer paper-based plate design.

Example 3

The selectivity of the assay on the paper-based well plate describedabove was evaluated by testing with several common anions and cations,including F⁻, I⁻, SCN⁻, CH₃COO⁻, NO₃ ⁻, C₂O₄ ²⁻, CO₃ ²⁻, SO₄ ²⁻, S²⁻,Mg²⁺, Al³⁺, Mn²⁺, Zn²⁺, Fe²⁺, Cd²⁺, Ni²⁺, Co²⁺, Hg²⁺, and Pb²⁺ at aconcentration of 1 mM. Their fluorescence signals were monitored andcompared with the fluorescence obtained from cyanide at 100 μM. Theresults showed in FIG. 20A and FIG. 20B that all tested anions andcations did not generate increasing signal. Hence, the sensor probe washighly selective toward cyanide.

According to the EPA standard method 335.4, the detection methoddescribed here, was intended to determine free cyanide in a solution. Ifthere is a presence of interferences in a solution, namely chlorine andsulfide, which can degrade cyanide into other forms, the sensor probecannot be used in this case.

The highly selective assay of copper-modulated chitosan-QDs wastransferred to the paper-based format presenting several advantages overa solution-based assay. One benefit was that paper-based well platesoffered higher sensitivity than the solution based formats. One example(where results are shown in FIGS. 21A and 21B), illustrated that that adried assay on a paper-based well plate has about 20 times highersensitivity than the assay in a solution due to the high surface tovolume ratio of GF/B used as a supporting material. QD sensors werepre-concentrated on the top surface of GF/B.

In order to maximize the sensitivity of the paper-based well platecyanide detection, the assay chemistry was optimized, including probeconcentration, amount of Cu²⁺ for quenching, and reaction time. Firstly,CS-QD520 concentration was varied from 4.08-20.43 μM. The calibrationgraphs in FIG. 22A showed that the calibration slopes dramaticallyincreased from 4.08 μM to 8.17 μM and after 8.17 μM, the slope did notsignificantly changed. The optimum sensor probe concentration was thenchosen at 8.17 μM. Second, the amount of Cu²⁺ to modulate thefluorescence signal played a crucial role for the sensitivity since thedetection mechanism was based on the formation of [Cu(CN)_(x)]^(1-x)species (see FIG. 22B). For example, if there is free Cu²⁺ in the assay,it will first form a complex with the added cyanide and no fluorescenceis generated. On the contrary, if there is less Cu²⁺ in the assay, highfluorescence background will be obtained, which then deteriorate thedetection limit. The optimum amount of Cu²⁺ (100 mg/L) for QD quenchingwas 40 μL per 1 mL of 8.17 μM CS-QD520. Third, the reaction time forfluorescence recovery was monitored, shown in FIG. 22C. After theintroduction of cyanide, fluorescence slowly regenerated owing to thereleasing of free CS-QD520 from copper. The longer reaction time, themore sensitive the assay. However, there was a necessity for minimizingreaction time while maintaining high sensitivity. The optimum reactiontime was chosen at 30 min because it gave higher sensitivity with lowstandard deviation.

A calibration curve for cyanide detection in water samples wasestablished with the optimum conditions. The data for calibration curveswas collected both within-day and between-days (4 days; n=12). A linearcalibration plot in a range of 0-200 μM was generated from the entiredata pool; y=27.441x+1398 (R²=0.997) (FIG. 23).

Limit of detection (LOD) and limit of quantification (LOQ) weredetermined using the International Union of Pure and Applied Chemistry(IUPAC) definitions. LOD and LOQ were 11.6 μM (0.3 μg/mL) and 38.7 μM (1μg/mL), respectively. The LOD presented here by disposable paper-basedwell plate was comparable to the commercially available, “Cyantesmo kit”(0.25-30 μg/mL) with quantitative results.

The real sample analysis was performed by using drinking water as amatrix. Drinking water was obtained from a local store; adjusted to pHabove 12 with 50% NaOH and analyzed using the assay. It was found thatthere was no cyanide detected in the drinking water used. The accuracyand precision of the detection system were assessed by measuringrecovery at three standardized cyanide concentration levels added todrinking water; low (50 μM), medium (100 μM), and high (200 μM) over thecalibration curve (Table 1). At low cyanide concentration (50 μM), the %RSD was larger than that of high cyanide concentration (200 μM). Thedisposable paper-based well plate demonstrated high accuracy withacceptable recovery (the recovery levels recommended by the Associationof Official Analytical Chemists (AOAC) International are in the range of75-120% at the 1 ppm concentration level). The lower recovery of cyanideat 50 μM may result from the trace metal containing in the drinkingwater, which depleted the fortified cyanide standard.

Found % Recovery Add (μm) Value (μm) SD RSD Value SD 50 37.64 5.33 14.1675.28 10.66 100 82.72 8.75 10.57 82.72 8.75 200 198.25 12.44 6.27 99.136.22

Example 4

In this example, a paper-based well plate analytical device wasfabricated by aerosolized deposition of a polymer solution (7.5% (w.v.)PCL in toluene) onto a masked substrate. Whatman no. 1 CHR filter paperwas masked with Scotch Blue with Edgelock painters tape. A 96-well plateconfiguration was then designed in CAD software (Solidworks) andtransferred to the masked substrate with a suitable method, such as alaser cutter or plotting cutter. The prepared substrate was then treatedwith aerosolized PCL via an airbrush rendering the unmasked materialhydrophobic. The treated substrate was then dried under ambientconditions before the masking material was removed exposing thehydrophilic 96-well plate. A representative embodiment is illustrated inFIG. 24 and FIGS. 25A and 25B show the results obtained using the deviceof FIG. 24 in uric acid, glucose, and bilirubin (×2) assays.

Example 5

In this example, a paper-based well plate analytical device useful forfluorescence-based analytical techniques was prepared. The device wasfabricated by aerosolized deposition of a polymer solution (7.5% (w/v)PCL (MW=37,000) in toluene) onto a masked substrate. Ahlstrom Grade 8613black filter paper was masked with masking tape (namely, Scotch Bluewith Edgelock painters tape). The desired design (e.g., 96 well plate ormicrofluidic architecture) was then designed in CAD software(Solidworks) and transferred to the masked substrate with a lasercutter. The prepared substrate was then treated with aerosolized PCL viaan airbrush rendering the unmasked material hydrophobic. The treatedsubstrate was then dried under ambient conditions before the maskingmaterial was removed exposing the hydrophilic 96-well plate. The darkcolor of the filter paper significantly reduced the backgroundfluorescence associated with PCL and plain white filter paper.

Example 6

In this example, a low-volume paper-based well plate “dimple plate” wasfabricated by saturating porous media with PCL, followed by cut andstack lamination. Whatman no 1 CHR filter paper was saturated with asolution of PCL (15% (w/v) (MW=80,000) in toluene and hung to dry underambient conditions. The treated paper was hung to allow even evaporationfrom all sides of the substrate leading to an even distribution ofpolymer on all surfaces. Next, the well plate design (drawn in CADsoftware) was cut into the treated paper and the substrates (solidbottom and well plate top piece) were laminated at a temperature greaterthan 60° C. (using a Scotch TL901 thermal laminator on 5 mil setting˜120° C.). The plate is ready to use as a low volume well plate forcolorimetric and UV-Vis based assays. This saturation method also can beused to construct devices made of substrates permeated with PCL, such asthe well plate devices illustrated in FIGS. 26A and 26B.

Example 7

In this example, a low-volume paper-based well plate “dimple plate” forfluorescence-based analysis was fabricated by saturating porous mediawith PCL followed by cut and stack lamination. Ahlstrom Grade 8613 blackfilter paper was saturated with a solution of PCL (15% (w/v) (MW=80,000)in toluene and hung to dry under ambient conditions. The treated paperwas hung to allow even evaporation from all sides of the substrateleading to an even distribution of polymer on all surfaces. Next, thewell plate design (drawn in CAD software) was cut into the treated paperand the substrates (solid bottom and well plate top piece) werelaminated at a temperature greater than 60° C. (using a Scotch TL901thermal laminator on 5 mil setting ˜120° C.). The dark color of theplate reduces background fluorescence by limiting reflection from thewhite surface of most filter papers.

Example 8

In this example, a low-volume polymer-based well plate was fabricated byapplying an even layer of PCL on the surface of a polymer film followedby cut and stack lamination. PET sheets (3M high temperature copiertransparency sheets) were coated with an even layer of PCL ((7.5% w/v)(MW=37,000) in toluene with an airbrush and allowed to dry under ambientconditions. Next, the well plate design (drawn in CAD software) was cutinto the treated PET sheet and the substrates (solid bottom and wellplate top piece) were laminated at a temperature greater than 60° C.(using a Scotch TL901 thermal laminator on 5 mil setting ˜120° C.). Thewell plate is then ready to be used for colorimetric and UV-Vis assaysin either top or bottom read devices.

Example 9

In this example, a low-volume polymer-based well plate alternative isfabricated by applying an even layer of laser jet toner on the surfaceof a polymer film, and then cut and stack lamination. PET sheets (3Mhigh temperature copier transparency sheets) are coated with a singlelayer of black laser jet toner from an HP Laserjet color 500 M551printer. Then, the well plate design (drawn in CAD software) is cut intothe prepared PET sheet and the substrates (solid bottom and well platetop piece) are laminated with a Scotch TL901 thermal laminator on 5 milsetting (˜120° C.). The well plate is then ready to be used forcolorimetric and UV-Vis assays in either top or bottom read devices. Inthis embodiment, the toner can be used as an adhesive, but in otherembodiments, a hydrophobic polymer component can be used as an adhesive.A representative embodiment of a low-volume well plate embodiment isshown in FIGS. 27A-27E.

Example 10

Paper-based hybrid microfluidic well plates combine features ofpaper-based well plates and low volume paper-based well plates. Hybridmicrofluidic plates offer the ability to incorporate multiple materialssuch as filtration within the structure of a single plate. These devicesare fabricated by saturation porous media such as Whatman no. 1 CHR withPCL (15% (w/v) MW=80,000 in toluene or other suitable solvent) and hungto dry under ambient conditions. Hanging allows even evaporation of thetoluene leading to an even distribution of polymer throughout the mediaand on the surface. Microfluidic architecture is then cut into thematerial with a laser cutter (or cutting plotter). Porous membranematerial (matching the design cut into the PCL treated paper, thusproviding a sensor layer) is then cut using the same method. The finaldevice is assembled by lamination of the substrates, including theporous membrane, at a temperature greater than 60° C. A representativeembodiment is illustrated in FIGS. 6, 7, and 8.

Example 11

A reusable frame for use with all well plate based devices wasfabricated via 3D printing. The frame was divided into two components, asecond piece matching the dimensions of standard well plates, and afirst piece for clamping well plate based devices. Both of thefabricated pieces were first designed in Solidworks (CAD software) andthen printed with an Ultimaker 2 3D printer using polylactic acid (PLA),a biodegradable material. Neodymium magnets were then embedded in bothportions of the frame (6 per section) allowing strong clamping andself-alignment of the frame pieces around well plate based devices. Analignment device was also fabricated in the same manner. The alignmentdevice is composed of a rectangular frame that fits around the exteriorof the second well plate frame ensuring reproducible alignment of thewell plate based devices. Proper alignment facilitates use with currenttechnologies, such as plate readers and liquid handling instrumentationthat are calibrated for standard 96 well plates.

Example 12

In this example, a valve-containing embodiment is described. Asillustrated in FIG. 28, valve-containing device 2800 comprises fourlayers, including a bottom layer 2802, an intermediate substrate 2804,sensor substrates 2806, and a top substrate 2808. Top substrate 2808comprises channels 2810, which comprise a polymer capable of beingmelted such that it serves as valve controlling flow between openings2812 of the top substrate. By irradiating channels 2810 of the topsubstrate 2808, the polymer in the channels can be melted and flow canoccur through the otherwise blocked channel. An exemplary schematicshowing how the valves work, such as the valves illustrated in FIG. 28,are provided by FIGS. 29A and 29B. As illustrated in FIG. 29A, which isa cross-sectional view of a device 2900, regions of a top layer 2902 canbe filled or saturated with a low melting material 2904. The low meltingmaterial 2904 can be positioned above sensor substrate layer 2906, whichis adjacent to a bottom substrate 2908, and comprises hydrophilicconnections 2910 between each well region 2912 of the sensor substrate.Upon applying localized heat (represented by arrow 2914), the lowmelting material 2904 is melted and can move into the hydrophilicconnections 2910 so as to block flow between the well regions 2912. Inanother embodiment illustrated in FIG. 29B, a sump component 2916 can beused in bottom substrate 2908 so as to provide a region into which a lowmelting component 2904, which is positioned between well regions 2912,can be received after melting. By doing so, the previously blockedhydrophilic connection 2910 is opened such that flow between wellregions 2912 can occur.

Example 13

FIG. 30 is an image of a device having wells that have been modifiedwith bovine serum albumin. As shown by FIG. 30, there is limitedinteraction between the wells of the substrate and an aqueous blue dyedeposited within the well.

Example 14

In this example, a surface-passivated intermediate substrate (shown byFIG. 31) is configured to comprise three different wells: (a) a firstwell 3100 that has been surface-passivated with bovine serum albumin;(b) a second well 3102 that was not surface-passivated; and (c) a thirdwell 3104 treated with a TMCS coupling reagent. The device was exposedto an assay component, EvaGreen dye, which is used for DNAquantification. As shown by FIG. 31, the activity of the EvaGreen dyeimmobilized in the first and third wells comprising thesurface-passivating reagent and the TMCS, respectively, was preserved,whereas the second well, which did not comprise a surface passivatingagent, did not preserve the dye's activity as evidenced by visualizationof the dye (suggesting the denaturing of the dye due to the surfacedeposition).

VII. Overview of Several Embodiments

Described herein are embodiments of polymer-based analytical devices,comprising: a substrate comprising a coating of a hydrophobic polymercomponent wherein the coating of the hydrophobic polymer is configuredto define outer perimeters of wells on or openings in the substrate andwherein the hydrophobic polymer component has a structure satisfyingFormula I

wherein Z, Y, and W independently can be O, S, NH, or NR², where R² ishydrogen, aliphatic, aryl, or heteroaryl; each of R³, R⁴, R⁵ and R⁶ (ifpresent) independently are hydrogen, aliphatic, aryl, heteroaryl, or aheteroatom-containing moiety; r is an integer selected from 1 to 4; sand t independently are integers selected from 0 to 4; and q is aninteger selected from 1 to 1000.

In any or all of the above embodiments, the substrate is a papersubstrate.

In any or all of the above embodiments, the substrate is a black papersubstrate.

In any or all of the above embodiments, the substrate further comprisesa fluidic channel formed in the coating of the hydrophobic polymercomponent, wherein the fluidic channel fluidly connects two or morewells or openings of the substrate.

In any or all of the above embodiments, the substrate is treated with asurface passivating agent. In some embodiments, the passivating agent isa blocking agent selected from bovine serum albumin or milk protein; ora silylating agent.

In any or all of the above embodiments, the substrate comprising acoating of a hydrophobic polymer component can be a top substrate andthe polymer-based analytical device can further comprise: anintermediate substrate coupled to the bottom substrate, the intermediatesubstrate comprising a coating of a hydrophobic polymer component thatdefines outer perimeters of wells on or openings in the intermediatesubstrate and having a pattern matching a pattern of the wells oropenings of the intermediate substrate; a sensor substrate coupled tothe intermediate substrate, the sensor substrate comprising a signalingmoiety or a sample; and a bottom substrate coated with a hydrophobicpolymer; wherein the hydrophobic polymer component of the top substrate,the intermediate substrate, and the bottom substrate independently has astructure satisfying Formula I:

wherein Z, Y, and W independently can be O, S, NH, or NR², where R² ishydrogen, aliphatic, aryl, or heteroaryl; each of R³, R⁴, R⁵ and R⁶ (ifpresent) independently are hydrogen, aliphatic, aryl, heteroaryl, or aheteroatom-containing moiety; r is an integer selected from 1 to 4; sand t independently are integers selected from 0 to 4; and q is aninteger selected from 1 to 1000.

In yet additional embodiments, the polymer-based analytical devicecomprises a bottom substrate comprising a coating of a hydrophobicpolymer component; an intermediate substrate coupled to the bottomsubstrate, the intermediate substrate comprising a coating of ahydrophobic polymer component that defines outer perimeters of wells onor openings in the intermediate substrate; a sensor substrate coupled tothe intermediate substrate, the sensor substrate comprising a signalingmoiety or a sample; and a top substrate coupled to the sensor substrate,the top substrate comprising a coating of a hydrophobic polymercomponent that defines outer perimeters of wells or openings having apattern matching a pattern of the wells or openings of the intermediatesubstrate; wherein the hydrophobic polymer component has a structuresatisfying Formula I:

wherein Z, Y, and W independently can be O, S, NH, or NR², where R² ishydrogen, aliphatic, aryl, or heteroaryl; each of R³, R⁴, R⁵ and R⁶ (ifpresent) independently are hydrogen, aliphatic, aryl, heteroaryl, or aheteroatom-containing moiety; r is an integer selected from 1 to 4; sand t independently are integers selected from 0 to 4; and q is aninteger selected from 1 to 1000.

In any or all of the above embodiments, the bottom substrate, theintermediate substrate and the top substrate are made of a cellulosicmaterial.

In any or all of the above embodiments, the sensor substrate comprises asignaling moiety capable of producing a fluorescent or colorimetricsignal.

In any or all of the above embodiments, the hydrophobic polymercomponent is polycaprolactone, polycaprolactone diol, polycaprolactonetriol, polycaprolactone-block-polytetrahydrofuan-block polycaprolactone,poly(ethylene oxide)-block-polycaprolactone, poly(ethyleneglycol)-block-poly(e-caprolactone) methyl ether, or any combinationthereof.

In any or all of the above embodiments, the bottom substrate, theintermediate substrate and the top substrate are black paper substrates.

In any or all of the above embodiments, the sensor layer comprises aplurality of individual sensor substrates configured to have dimensionsmatching dimension of the wells or openings of the intermediatesubstrate.

In any or all of the above embodiments, the intermediate substratefurther comprises a fluidic channel formed in the coating of thehydrophobic polymer component, wherein the fluidic channel fluidlyconnects two or more wells or openings of the intermediate substrate.

Also disclosed herein are embodiments of methods for makingpolymer-based analytical devices, comprising: masking a substrate madeof a polymeric material with a masking material to form a maskedsubstrate; patterning the masked substrate by cutting a pre-determinedpattern into the masking material thereby providing hydrophilic unmaskedareas of the masked substrate and masked areas of the masked substrate;coating the hydrophilic unmasked areas of the substrate with ahydrophobic polymer layer thereby converting the hydrophilic unmaskedareas of the masked substrate to hydrophobic unmasked areas; andremoving any remaining masking material from the masked substrate toexpose one or more wells or openings, each of which has an outerperimeter defined by the hydrophobic polymer layer of the substrate andwherein the wells or openings are configured in the pre-determinedpattern.

In any or all of the above embodiments, the method can further comprisecombining the substrate with one or more additional hydrophobicpolymer-coated substrates.

In any or all of the above embodiments, the method can further compriselaminating the substrate and the one or more additional substratestogether such that the hydrophobic polymer layer of the one or moresubstrates bonds the substrate and the one or more additional substratestogether.

In additional embodiments, the methods can comprise making asubstrate-based analytical device. Such methods can comprise exposing apolymeric substrate to a solution of a hydrophobic polymer component toform a layer of the hydrophobic polymer component that fully covers thepolymeric substrate thereby forming a fully-coated polymeric substrate;patterning the fully-coated polymeric substrate to comprise a pluralityof wells or openings thereby forming a patterned polymeric substrate,wherein each well or opening of the plurality of wells or openings hasan outer perimeter defined by the hydrophobic polymer; exposing thepatterned polymeric substrate to O₂ to render the hydrophobic polymerlocated in the wells or openings; and coupling the patterned polymericsubstrate with a hydrophobic polymer-coated bottom substrate.

In any or all of the above embodiments, coupling comprises laminatingthe polymer-coated bottom substrate and the patterned polymericsubstrate.

In any or all of the above embodiments, the polymeric substrate is apaper substrate or a transparent polymer-based substrate.

In some embodiments, the method of making device embodiments describedherein can comprise making the intermediate substrate and the topsubstrate using a screen printing technique whereby the hydrophobicpolymer component is screen printed onto the intermediate substrate andthe top substrate.

Also disclosed herein are embodiments of a plate frame, comprising: afirst component made of a biodegradable polymeric material and having anouter perimeter section; a second component made of a biodegradablepolymeric material and having an outer perimeter section configured toalign with the outer perimeter section of the first component; one ormore magnets positioned within the outer perimeter section of the secondcomponent; and one or more magnets positioned within the outer perimetersection of the first component, which magnets of the first component arepositioned to align with the magnets of the second component and securethe first component to the second component in a predetermined alignmentby magnetic attraction; wherein when the first component and the secondcomponent are associated together, they define a chamber that isconfigured to receive the polymer-based analytical device of any or allof the above embodiments between the second component and the firstcomponent.

In view of the many possible embodiments to which the principles of thepresent disclosure may be applied, it should be recognized that theillustrated embodiments are only examples and should not be taken aslimiting the scope of the present disclosure. Rather, the scope isdefined by the following claims. We therefore claim as our invention allthat comes within the scope and spirit of these claims.

We claim:
 1. A polymer-based analytical device, comprising: a substratecomprising a coating of a hydrophobic polymer component wherein thecoating of the hydrophobic polymer is configured to define outerperimeters of wells on or openings in the substrate and wherein thehydrophobic polymer component has a structure satisfying Formula I

wherein Z, Y, and W independently can be O, S, NH, or NR², where R² ishydrogen, aliphatic, aryl, or heteroaryl; each of R³, R⁴, R⁵ and R⁶ (ifpresent) independently are hydrogen, aliphatic, aryl, heteroaryl, or aheteroatom-containing moiety; r is an integer selected from 1 to 4; sand t independently are integers selected from 0 to 4; and q is aninteger selected from 1 to
 1000. 2. The polymer-based analytical deviceof claim 1, wherein the substrate is a porous substrate.
 3. Thepolymer-based analytical device of claim 1, wherein the substrate is ablack paper substrate or a glass microfiber substrate.
 4. Thepolymer-based analytical device of claim 1, wherein the substratefurther comprises a fluidic channel formed in the coating of thehydrophobic polymer component, wherein the fluidic channel fluidlyconnects two or more wells or openings of the substrate.
 5. Thepolymer-based analytical device of claim 1, wherein the substrate istreated with a surface passivating agent.
 6. The polymer-basedanalytical device of claim 5, wherein the passivating agent is ablocking agent selected from bovine serum albumin or milk protein; or asilylating agent.
 7. The polymer-based analytical device of claim 1,wherein the hydrophobic polymer component is polycaprolactone,polycaprolactone diol, polycaprolactone triol,polycaprolactone-block-polytetrahydrofuan-block polycaprolactone,poly(ethylene oxide)-block-polycaprolactone, poly(ethyleneglycol)-block-poly(e-caprolactone) methyl ether, or any combinationthereof.
 8. The polymer-based analytical device of claim 1, wherein thesubstrate comprising a coating of a hydrophobic polymer component is atop substrate and the polymer-based analytical device further comprises:an intermediate substrate coupled to the bottom substrate, theintermediate substrate comprising a coating of a hydrophobic polymercomponent that defines outer perimeters of wells on or openings in theintermediate substrate and having a pattern matching a pattern of thewells or openings of the intermediate substrate; a sensor substratecoupled to the intermediate substrate, the sensor substrate comprising asignaling moiety or a sample; and a bottom substrate coated with ahydrophobic polymer; wherein the hydrophobic polymer component of thetop substrate, the intermediate substrate, and the bottom substrateindependently has a structure satisfying Formula I:

wherein Z, Y, and W independently can be O, S, NH, or NR², where R² ishydrogen, aliphatic, aryl, or heteroaryl; each of R³, R⁴, R⁵ and R⁶ (ifpresent) independently are hydrogen, aliphatic, aryl, heteroaryl, or aheteroatom-containing moiety; r is an integer selected from 1 to 4; sand t independently are integers selected from 0 to 4; and q is aninteger selected from 1 to
 1000. 9. The polymer-based analytical deviceof claim 8, wherein the bottom substrate, the intermediate substrate,and the top substrate are made of a cellulosic material.
 10. Thepolymer-based analytical device of claim 8, wherein the sensor substratecomprises a signaling moiety capable of producing a fluorescent orcolorimetric signal.
 11. The polymer-based analytical device of claim 8,wherein the bottom substrate, the intermediate substrate, and the topsubstrate are black paper substrates.
 12. The polymer-based analyticaldevice of claim 8, wherein the sensor substrate comprises a plurality ofindividual sensor substrates configured to have dimensions matchingdimension of the wells or openings of the intermediate substrate. 13.The polymer-based analytical device of claim 8, wherein the intermediatesubstrate further comprises a fluidic channel formed in the coating ofthe hydrophobic polymer component, wherein the fluidic channel fluidlyconnects two or more wells or openings of the intermediate substrate.14. A method, comprising: masking a substrate made of a polymericmaterial with a masking material to form a masked substrate; patterningthe masked substrate by cutting a pre-determined pattern into themasking material thereby providing hydrophilic unmasked areas of themasked substrate and masked areas of the masked substrate; coating thehydrophilic unmasked areas of the substrate with a hydrophobic polymerlayer thereby converting the hydrophilic unmasked areas of the maskedsubstrate to hydrophobic unmasked areas; and removing any remainingmasking material from the masked substrate to expose one or more wellsor openings, each of which has an outer perimeter defined by thehydrophobic polymer layer of the substrate and wherein the wells oropenings are configured in the pre-determined pattern, thereby providinga polymer-based analytical device.
 15. The method of claim 14, furthercomprising combining the substrate with one or more additionalhydrophobic polymer-coated substrates.
 16. The method of claim 14,further comprising laminating the substrate and the one or moreadditional substrates together such that the hydrophobic polymer layerof the one or more substrates bonds the substrate and the one or moreadditional substrates together.
 17. A method of making a substrate-basedanalytical device, comprising: exposing a polymeric substrate to asolution of a hydrophobic polymer component to form a layer of thehydrophobic polymer component that fully covers the polymeric substratethereby forming a fully-coated polymeric substrate; patterning thefully-coated polymeric substrate to comprise a plurality of wells oropenings thereby forming a patterned polymeric substrate, wherein eachwell or opening of the plurality of wells or openings has an outerperimeter defined by the hydrophobic polymer; exposing the patternedpolymeric substrate to O₂ to render the hydrophobic polymer located inthe wells or openings; and coupling the patterned polymeric substratewith a hydrophobic polymer-coated bottom substrate.
 18. The method ofclaim 17, wherein coupling comprises laminating the polymer-coatedbottom substrate and the patterned polymeric substrate.
 19. The methodof claim 17, wherein the polymeric substrate is a paper substrate or atransparent polymer-based substrate.
 20. A method of making the deviceof claim 8, wherein the intermediate substrate and the top substrate aremade by screen printing the hydrophobic polymer component onto theintermediate substrate and the top substrate.
 21. A plate frame,comprising: a first component made of a polymeric material and having anouter perimeter section; a second component made of a polymeric materialand having an outer perimeter section configured to align with the outerperimeter section of the first component; one or more magnets positionedwithin the outer perimeter section of the second component; and one ormore magnets positioned within the outer perimeter section of the firstcomponent, which magnets of the first component are positioned to alignwith the magnets of the second component and secure the first componentto the second component in a predetermined alignment by magneticattraction; wherein when the first component and the second componentare associated together, they define a chamber that is configured toreceive the polymer-based analytical device of claim 1 between thesecond component and the first component.